1. Field of the Invention
The present invention is broadly concerned with oligomers capable of inhibiting expression of collagen genes, and corresponding methods. More particularly, the invention pertains to transcription-inhibiting complexes, each composed of an oligomer bound to a polypurine-polypyrimidine region of a collagen-gene promoter region.
2. Description of the Prior Art
Collagen is a family of fibrous proteins present in all multicellular organisms. It is the major fibrous element of skin, bone, tendon, cartilage, blood vessels, and teeth, and is present in nearly all organs. Collagen is composed of a triple-helix formed from three polypeptide chains. These triple helices are wrapped around each other to form a super helix. In several types of collagen, including Type I collagen, these super helixes form fibrils. In the case of Type I collagen, the polypeptide triple helix is composed of two .alpha.1 polypeptide chains and one .alpha.2 polypeptide chain.
In response to tissue injury or invasion, a healing response is invoked that ultimately leads to an accumulation of fibrillar type I collagen. This is true for many systemic organs and the heart. Such a healing response, when unabated and invoked in the absence of injury, leads to a progressive interstitial fibrosis that proves pathologic. Parenchymal cell function is compromised by a disproportionate concentration of type I collagen, a characteristic feature of interstitial fibrosis in different organs (1-9). Various stages of organ dysfunction are marked by the activation and repression of type I collagen genes, thereby allowing for the design of specific agents to promote the necessary or adaptive phenotype or to repress the onset of pathologic interstitial fibrosis.
A wide array of hormones, cytokines, and growth factors have been implicated in the mediation of fibrous tissue formation (10-20). Many of these factors mediate their action through transcriptional mechanisms. Therefore, the study of transcriptional regulatory elements within the .alpha.1(I) and .alpha.2(I) collagen gene promoters and their transacting protein factors is of major interest. Effector cells which bring about fibrosis include interstitial fibroblasts and phenotypically transformed fibroblast-like cells termed myofibroblasts (21).
Several cis-acting elements in the .alpha.1(I) and .alpha.2(I) collagen genes located on both sides of the transcription start site as well as their transacting factors have been identified (for reviews, see 22-24). Very little is known about the factor(s) binding to the -200 to -140 region of the .alpha.1(I) collagen promoter. These sequences are highly conserved among mammals (25), and correspond to the DNase I hypersensitive regions around the transcriptional start site. It is generally believed that DNase I hypersensitivity represents nucleosome-free regions which can interact with various regulatory proteins (26). Cis-acting elements in the -190 to -170, and -160 to -133 regions of the mouse .alpha.1(I) promoter and trans-acting factors binding to these elements in NIH-3T3 fibroblast nuclear extracts have been studied in some detail (27). Competition experiments in EMSA's provided evidence that a single factor binds to both of these elements. Furthermore, in transient transfection experiments, while a three-bp substitution mutation in the more distal element (from -194 to -168) had little effect on the promoter activity, a three-bp mutation in the more proximal element (from -160 to -133) resulted in a four-fold increase in reporter gene expression, indicating that this factor negatively regulates transcription (designated IF-1 in 26). In contrast, Brenner et al. (28) have shown that deletion of both regions of the mouse .alpha.1(I) promoter resulted in decreased promoter activity implying positive activation of transcription.
Oligomers (i.e., oligonucleotides and oligonucleotide analogs such as protein nucleic acid) are reagents for inhibition of gene expression because of their high-affinity binding to specific nucleotide sequences. The best known strategy for oligomer reagents involves antisense oligonucleotides which bind mRNA to inhibit its processing or translation. For example, Laptev et al. (44) showed that the expression of the human .alpha.1(I) collagen gene is effectively inhibited by antisense oligonucleotides targeted at specific regions of the .alpha.1(I) mRNA. Additionally, gene promoters can serve as targets for a novel, antisense strategy, namely the triplex strategy (for reviews, see 40 and 45). This strategy employs single-stranded oligomers that bind to the major groove of a polypurine-polypyrimidine region of a double-stranded DNA to form a triple helix in a sequence-specific manner. These oligomers are called triplex-forming oligonucleotides (TFO's) or TFO analogs. In a polypurine-polypyrimidine region, a purine-rich DNA single strand is hydrogen bonded by Watson-Crick base-pairing to a pyrimidine-rich DNA single strand; the polypurine-polypyrimidine region is not necessarily a homopurine-homopyrimidine region in that the purine-rich DNA single strand may contain at least one pyrimidine residue and the pyrimidine-rich DNA single strand may contain at least one purine residue. These triplexes have been shown to inhibit sequence-specific DNA-binding proteins thereby affecting the transcriptional activity of various promoters in both in vitro and in vivo experiments (29-32). However, the use of oligomers to inhibit transcription of a collagen gene is unknown in the prior art.